Thanks for the advice Philip...we tried 10ul last night; I'll see how it goes. We originally thought the issue could be resolved by using enrichment broths, TB or 2xLB and/or increasing the incubation time from 22 to 24 then 26 hours. We use the Millipore Montage system, which in the past has given use good results, we start automating the process next week on Biomek NX's on 384-format for both the prep and sequence set-up & clean-up steps. That might take away some of the variability in the manual way we have been doing things of late...but it will probably just give me alot more to worry about.
One thing I forgot to ask was how do you normally QC your library transformations? We tend just to take about 24-36 random clones and either restrict or (less reliably) PCR check using the M13 primers. With all the transformations we've done this for we get good results i.e. high level of recombination & insert size...it just then doesn't follow when we ramp up the plasmid preps.
Ciaran
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